Development and application of stress-signal transduction system as an assessment ofbiomaterials

在三种MAP激酶级联反应中，其中两种集中于JNKs和p38 MAP激酶的级联反应优先被细胞毒性应激（如紫外线辐射、x射线、热休克和渗透休克）和促炎细胞因子（如肿瘤坏死因子（TNF）和白细胞介素-1）激活。通过这些应激激活的MAP激酶途径介导的重要生物学反应之一似乎是通过调节细胞凋亡来决定细胞命运。在本研究项目中，我们打算通过分析应激激活的MAP激酶通路的激活状态来开发一种新的、快速和敏感的生物材料评估系统。我们首先建立了INK和p38激活的灵敏检测系统，并清楚地表明凋亡信号调节激酶1 (ASK1)，已知可调节JNK和p38 MAP激酶途径，是氧化应激诱导JNK和p38激活所必需的。接下来，我们建立了ASK1激活的灵敏检测系统。我们发现ASK1的激酶结构域Thr845的磷酸化是ASK1激活所必需的，并提出了一种特异性识别ASK1的Thr845激活状态的多克隆抗体。该抗磷酸化askl抗体检测ASK1激活状态的灵敏度远高于常规的“体外激酶试验”。通过使用该抗体的各种生化分析和对askl缺陷小鼠的进一步研究，我们发现ASK1不仅是氧化应激所必需的，也是内质网络应激诱导的细胞凋亡所必需的，这表明ASK1- map激酶途径是各种应激源的共同信号介质。因此，准确和灵敏地检测ASK1-MAP激酶途径的激活状态似乎对我们的各种生物材料评估系统的开发和应用是有用的。

Among the three MAP kinase cascades, two of them that converge on JNKs and p38 MAP kinases are preferentially activated by cytotoxic stresses such as UV radiation, X-ray, heat shock and osmotic shock, and by proinflammatory cytokines such as tumor necrosis factor (TNF) and interleukin-1. One of the important biological responses mediated through these stress-activated MAP kinase pathways appears to be the decision of cell fate by regulating apoptosis. In this research project, we intended to develop a novel, rapid and sensitive assessment system for various biomaterials by assaying the activation status of the stress-activated MAP kinase pathways. We first established the sensitive assay system for INK and p38 activation, and clearly showed that apoptosis signal^egulating kinase 1 (ASK1), which is known to regulate the JNK and p38 MAP kinase pathways, is required for oxidative stress-nduced activation of JNK and p38. We next established the sensitive assay system for ASK1 activation. We found that phosphorylation of Thr845 in the kinase domain of ASK1 is required for the activation of ASK1, and raised a polyclonal antibody that specifically recognized the activation status of Thr 845 of ASK 1. This anti-phospho-ASKl antibody could detect the activation state of ASK1 with much higher sensitivity than the conventional "in vitro kinase assay". By the various biochemical analyzes using this antibody and the further investigation of ASKl-deficient mice, we have found that ASK1 is necessary for not only oxidative stress- but also ER stress-induced apoptosis, suggesting that the ASK1-MAP kinase pathways is a common signal mediator of various kind of stressors. Thus, accurate and sensitive detection of the activation status of the ASK1-MAP kinase pathways appears to be useful for the development and application of our assessment system for various biomaterials.